Mesoxin-producing strain and application thereof

Abstract

This invention relates to a streptomycin-producing strain and its application, wherein the strain is Streptomyces. Streptomyces sp. ZS-1 has good activity in producing kasugamycin, and its fermentation products have higher antibacterial activity and a wider antibacterial range.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural technology, and particularly relates to a mesoxin production strain and application thereof. BACKGROUND

[0002] Mesoxin is an agricultural antibiotic bactericide independently developed by China, which can inhibit protein synthesis of pathogenic bacteria, has low toxicity and low residue, and can be used to prevent and treat various crop diseases.

[0003] Mesoxin is a multi-component mixture produced by fermentation of Streptomyces, and the effective components mainly include mesoxin A, B, C, D, E and F. Different strains actually produce different types of effective components and different proportions of each component, which will make the bacteriostatic ability different.

[0004] At present, obtaining a strain with high mesoxin yield and strong bacteriostatic ability is a key measure to realize pesticide efficiency increase and dosage reduction. SUMMARY

[0005] In order to solve the above technical problems, the present application provides a mesoxin production strain, which not only has good mesoxin production activity, but also has higher bacteriostatic activity and a wider bacteriostatic range of fermentation products.

[0006] According to one aspect of the present application, a mesoxin production strain is provided, and the strain is Streptomyces sp. ZS-1. Streptomyces The strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 35482.

[0007] According to one aspect of the present application, a mesoxin production strain is provided, and the strain is Streptomyces sp. ZS-1. Streptomyces The strain satisfies any one or a combination of the following conditions:

[0008] (1) contains a molecular marker, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1;

[0009] (2) contains a molecular marker, and the nucleotide sequence of the molecular marker has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1;

[0010] (3) the genomic DNA is amplified using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, and a specific band can be obtained by agarose gel electrophoresis analysis of the amplification product;

[0011] (4) using primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 to amplify the genomic DNA, sequencing the amplified product, and obtaining a nucleotide sequence shown in SEQ ID NO: 1; or

[0012] The obtained nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0013] According to an aspect of the present application, a molecular marker is provided, the nucleotide sequence of which is shown in SEQ ID NO: 1; or

[0014] The nucleotide sequence of the molecular marker has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0015] According to an aspect of the present application, the use of the above-mentioned molecular marker in identifying the above-mentioned strain is provided.

[0016] According to an aspect of the present application, a primer pair is provided, the nucleotide sequence of the forward primer is shown in SEQ ID NO: 2; and

[0017] the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 3.

[0018] According to an aspect of the present application, the use of the above-mentioned primer pair in identifying the above-mentioned strain is provided.

[0019] According to an aspect of the present application, a method for identifying the above-mentioned strain is provided, comprising the following steps:

[0020] (1) sequencing the strain;

[0021] (2) obtaining a sequence containing a specific molecular marker, the specific molecular marker satisfying any one or a combination of the following:

[0022] 1) the nucleotide sequence is shown in SEQ ID NO: 1; and / or

[0023] 2) the nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0024] Optionally, the sequencing is selected from:

[0025] (1) genomic sequencing; and / or

[0026] (2) PCR product sequencing.

[0027] Optionally, the PCR product is obtained by amplification using primers shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0028] According to an aspect of the present application, there is provided a use of the above-mentioned strain in the preparation of a pesticide product.

[0029] According to an aspect of the present application, there is provided a pesticide product containing a fermentation product of the above-mentioned strain, wherein the effective component of the fermentation product contains mesoxin.

[0030] Optionally, the pesticide product is selected from any one of a fermentation stock solution, a fermentation dilute solution, a fermentation concentrate solution, a technical material, a mother material, a preparation.

[0031] Optionally, the mesoxin content of the fermentation stock solution, the fermentation dilute solution or the fermentation concentrate solution is 10-300000 μg / mL.

[0032] The mesoxin content of the technical material or the mother material is 5-99%.

[0033] The mesoxin content of the preparation is 0.1-30%.

[0034] Optionally, the dosage form of the preparation is selected from any one of a powder, a granule, an oil, a seed treatment suspension.

[0035] Optionally, the dosage form of the preparation is selected from a sustained release agent.

[0036] Optionally, the dosage form of the preparation is selected from a suspension.

[0037] Optionally, the dosage form of the preparation is selected from a suspension seed coating agent.

[0038] Optionally, the dosage form of the preparation is selected from any one of a seed treatment dispersible powder, a seed treatment soluble powder, a seed treatment liquid, a seed treatment emulsion, a seed treatment microcapsule suspension.

[0039] Optionally, the dosage form of the preparation is selected from any one of a large granule, a fine granule, a microgranule, a microcapsule granule, a wettable powder, an oil dispersible powder, a water dispersible granule, a cream granule, an effervescent granule, a dispersible tablet, an effervescent tablet, a sustained release block, a sustained release tube, a sustained release granule, a soluble powder, a soluble granule, a soluble tablet, a soluble liquid, a water agent, a soluble gel agent, an expanded film oil agent, an ultra-low volume liquid agent, an ultra-low volume microcapsule suspension, an emulsion oil agent, an emulsion, a dispersible liquid agent, a paste, a concentrated gum agent, a water emulsion agent, an oil emulsion agent, a microemulsion, a grease, a microcapsule suspension, an oil suspension, a suspension emulsion.

[0040] According to an aspect of the present application, a preparation method of the above-mentioned pesticide product is provided, and the preparation method comprises the following steps:

[0041] The fermentation is performed by using Streptomyces sp. ZS-1, and the pesticide product is obtained. Streptomyces

[0042] Optionally, the preparation method comprises the following steps:

[0043] (1) the fermentation is performed by using Streptomyces sp. ZS-1, and a fermentation stock solution, i.e. the pesticide product, is obtained; or Streptomyces

[0044] The preparation method further comprises the following steps:

[0045] (2) the fermentation stock solution is further processed, and any one of a fermentation diluent, a fermentation concentrate, a raw pesticide, a mother pesticide, and a preparation is obtained, i.e. the pesticide product.

[0046] According to an aspect of the present application, the above-mentioned strain, the above-mentioned pesticide product, or the pesticide product obtained according to the above-mentioned method is used for plant disease control.

[0047] According to an aspect of the present application, the above-mentioned strain, the above-mentioned pesticide product, or the pesticide product obtained according to the above-mentioned method is used for preparing a plant disease control agent.

[0048] The positive and beneficial effects achieved by the present application are as follows:

[0049] 1. The Streptomyces sp. ZS-1 provided in the present application has a high production activity of mesoxins, and the mass content and biological titer level of mesoxins in the fermentation liquor are significantly higher than those of existing strains. Streptomyces 2. The fermentation product of the Streptomyces sp. ZS-1 provided in the present application has higher bacteriostatic activity and a wider bacteriostatic range. Compared with existing strains, the strain provided in the present application exhibits stronger bacteriostatic effect.

[0050] Streptomyces 3. The Streptomyces sp. ZS-1 provided in the present application can inhibit not only bacterial pathogenic fungi but also fungal pathogenic fungi such as Sclerotium rolfsii, and has a wider bacteriostatic range.

[0051] Streptomyces

[0052] The Streptomyces sp. ZS-1 was deposited in the China General Microbiological Culture Collection Center on July 31, 2025, and was named Streptomyces sp. ZS-1. Streptomyces Streptomyces ​​​​​​sp., with accession number CGMCC No. 35482. Attached Figure Description

[0053] Figure 1 This is an image of the inhibition zones of 10 single colonies in dish 1 of Example 1;

[0054] Figure 2 Streptomyces Streptomyces Phylogenetic tree of sp. ZS-1 16S rDNA;

[0055] Figure 3 Streptomyces Streptomyces Liquid chromatography chromatogram of ZS-1 fermentation broth;

[0056] Figure 4 It is the Hainan variant of Streptomyces lilacinus ( Streptomyces lavendulae Liquid chromatography-mass spectra of UV-11 fermentation broth (var.hainanensis);

[0057] Figure 5 The mass spectrum of component 2 is shown below.

[0058] Figure 6 The H-NMR spectrum of component 2;

[0059] Figure 7 For component 2 13 C-NMR spectrum;

[0060] Figure 8 The mass spectrum of component 4 is shown below.

[0061] Figure 9 The H-NMR spectrum of component 4;

[0062] Figure 10 For component 4 13 C-NMR spectrum;

[0063] Figure 11 The antibacterial effects of various fermentation broths on peanut white mold pathogens were investigated.

[0064] Figure 12 The agarose gel electrophoresis results are for the amplification products of SEQ ID NO: 2 and SEQ ID NO: 3. Detailed Implementation

[0065] For the purposes of this invention, unless otherwise stated, the terms used in this application have the following meanings:

[0066] The term "active ingredient" refers to the component in pesticide products that has toxic and killing activity against diseases, insects, and weeds.

[0067] The term "technical material" means a product obtained in the course of production consisting of the active ingredient and the relevant impurities, to which a small amount of additives can be added, if necessary.

[0068] The term "technical material" means a product obtained in the course of production consisting of the active ingredient and the relevant impurities, to which a small amount of additives can be added, if necessary.

[0069] The term "formulation" means a product which is stable in its state, processed from a pesticide technical material (mother drug) and suitable adjuvants, or processed from biological fermentation, plant extraction, etc.

[0070] The term "plant disease" means that the growth and development of plants are significantly hindered due to the infection of other organisms and the influence of adverse non-biological factors, and pathological changes will occur in the interior and exterior of the plant, in the physiology and tissue structure, and the plant will appear sick and even die, resulting in reduced yield and poor quality. The "plant disease" of the present application specifically refers to the disease caused by the infection of other organisms on plants, which include but are not limited to fungi (such as Sclerotium rolfsii), or bacteria (such as Pseudomonas syringae pv. lachrymans), etc.

[0071] The reagents and solvents used in the following examples can be purchased by commercial channels, or prepared by conventional methods known to those skilled in the art. The experimental methods not specified in the following examples are carried out according to conventional methods and conditions.

[0072] Preparation of PDA medium: 200 g of potatoes were washed, peeled, and chopped, and then boiled in 800 ml of distilled water for half an hour. The mixture was filtered through gauze, and then 20 g of glucose and 20 g of agar were added. The volume was made up to 1000 ml with distilled water, and sterilized at 121°C for 30 minutes.

[0073] Preparation of LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 20 g of agar were dissolved and made up to 1000 ml with distilled water, and the pH was adjusted to 7.0. The mixture was sterilized at 121°C for 20 minutes.

[0074] Preparation of seed medium: the materials were weighed according to the following proportions: glucose 1.0%, corn starch 1.5%, corn flour 1.0%, soybean flour 2.0%, sodium chloride 0.3%, ammonium chloride 0.3%, calcium carbonate 0.3%, and the rest was water. The pH was adjusted to 6.6, and the mixture was sterilized at 121°C for 20 minutes.

[0075] Fermentation medium preparation: each material was weighed according to the following proportion, glucose 5.0%, corn starch 2.0%, corn flour 2.0%, soybean meal 4.0%, sodium chloride 0.6%, ammonium chloride 0.8%, calcium carbonate 0.7%, potassium dihydrogen phosphate 0.02%, magnesium sulfate 0.05%, and the rest was water, the pH was adjusted to 6.6, and sterilized at 121°C for 20 min.

[0076] The liquid phase detection method (HPLC) and the biological titer detection method refer to the enterprise standard Q / KLSW 043-2021 "80U / mg (6%) mesonitale soluble liquid" of Fujian Kaili Biological Products Co., Ltd.

[0077] Genomic DNA extraction was performed using the bacterial genomic DNA extraction kit of Tiangeng Bioscience Co., Ltd., and the extraction method referred to the instruction manual thereof.

[0078] Specific amplification was performed using the premixed Taq enzyme Premix Taq™ (TaKaRa Taq™ Version 2.0) of Takara Bio Inc., and the amplification system was as follows:

[0079]

[0080] The amplification program was as follows:

[0081]

[0082] The source of the Streptomyces used in the present application is explained as follows:

[0083]

[0084] Example 1 Streptomyces Streptomyces Isolation and identification of strain ZS-1

[0085] 1. Strain isolation

[0086] Ten soil samples were taken from Sanjiangkou Hot Spring Wetland Park in Haicang District, Xiamen City, Fujian Province, each weighing 10 g, dissolved in 90 mL of sterile water, and gradient diluted with sterile water to 100 times and 1000 times, and an appropriate amount of 1000 times dilution was spread on PDA culture medium plates, and cultured at 28°C for 3 days. 25 single colonies that grew out were further purified and cultured, and the purified single colonies were inoculated into triangular flasks containing fermentation medium, and fermented at a temperature of 28°C and a rotation speed of 180 rpm for 3 days to obtain fermentation broth. The fermentation broth was filtered through a 0.22 μm bacterial filter to remove the bacterial cells, and was ready for use.

[0087] Take 200 mL of thawed and cooled LB medium to 48 ℃~50 ℃, and add 2 mL of 1×10 8 A CFU / mL suspension of *Cucumber angular leaf spot* was prepared by shaking and pouring an appropriate amount into a 15cm diameter petri dish. The mixture was allowed to solidify to obtain a bacterial layer. Three bacterial layers were prepared, designated as dishes 1-3. Ten filter paper discs were placed in dishes 1 and 2, and five in dish 3. 20μL of the fermentation broth containing 25 single colonies was added to each filter paper disc. The discs were then dried and incubated at 28℃ for one day. The size of the inhibition zone was observed, and the strain with the largest inhibition zone (strain number 9 in dish 1) was selected. The inhibition effect is shown in [reference needed]. Figure 1 Further research will be conducted.

[0088] 2. Strain identification

[0089] The above-mentioned strain 9 was identified using the 16S rDNA identification method. The specific identification steps are as follows:

[0090] (1) Genomic DNA extraction: After activating and culturing strain 9 on PDA medium plates, genomic DNA was extracted.

[0091] (2) 16S rDNA amplification: Using genomic DNA as a template, 16S rDNA fragments were amplified from genomic DNA using universal 16S rDNA primers (27F sequence as shown in SEQ ID NO: 4 and 1492R sequence as shown in SEQ ID NO: 5). The amplification products were sent to Beijing Liuhe BGI Genomics Co., Ltd. for analysis.

[0092] (3) 16S rDNA sequence analysis: The 16S rDNA sequence obtained from sequencing was compared with the 16S rDNA sequence in GenBank using BLAST. The results showed that it was consistent with... Streptomyces The 16S rDNA sequence of sp. strain SK60 showed the highest similarity (>99%). A phylogenetic tree was constructed using Mega 5.0 software, and this strain was related to... Streptomyces sp. strainSK60 clusters on one branch (e.g.) Figure 2 (As shown).

[0093] Based on the above molecular identification results, the strain was identified as belonging to the genus Streptomyces. Streptomyces sp., named Streptomyces Streptomyces sp. ZS-1 is deposited at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35482.

[0094] Example 2 StreptomycesStreptomyces sp. ZS-1 active substance identification

[0095] 1. Liquid chromatography detection

[0096] The components of Streptomyces were located using kasugamycin B, kasugamycin C, kasugamycin D, kasugamycin E, and kasugamycin F standards (these standards are owned by Yuanda Crop Science (Shaanxi) Co., Ltd.). Streptomyces The fermentation broth of sp. ZS-1 was analyzed by liquid chromatography, and the chromatogram is shown below. Figure 3 As shown.

[0097] Based on the positioning of the streptomycin standard, the results showed that Streptomyces Streptomyces In the fermentation broth of sp. ZS-1, the component with a peak time of 26.556 (component 1) is kasugamycin B, the component with a peak time of 23.023 (component 2) is kasugamycin C, the component with a peak time of 17.823 (component 3) is kasugamycin D, the component with a peak time of 14.999 (component 4) is kasugamycin E, and the component with a peak time of 12.126 (component 5) is kasugamycin F.

[0098] Take Streptomyces hainanensis (a type of Streptomyces lilacinus) Streptomyces lavendulae The fermentation broth of var. hainanensis (UV-11) was analyzed using kasugamycin B, C, D, E, and F standards to locate each component. Liquid chromatography was then performed on the fermentation broth, and the resulting chromatograms are shown below. Figure 4 As shown, the component with a peak time of 23.400 is kasugamycin B, the component with a peak time of 19.839 is kasugamycin C, the component with a peak time of 17.985 is kasugamycin D, the component with a peak time of 15.050 is kasugamycin E, and the component with a peak time of 12.155 is kasugamycin F.

[0099] The above results indicate that Streptomyces Streptomyces sp. ZS-1 and Streptomyces lilacinus Hainan variant ( Streptomyces lavendulae Both *var. hainanensis* and UV-11 can produce kasugamycin, but comparison shows that... Figure 3 and Figure 4 It was found that there were significant differences in the content and ratio of the various components of kasugamycin BF between the two strains.

[0100] 2. Structural identification

[0101] To further confirm the above-mentioned Streptomyces StreptomycesWhether components 1-5 of sp. ZS-1 are streptavidin B, C, D, E, and F was determined by gel column chromatography, concentration, and drying from Streptomyces. Streptomyces The above components were obtained from the fermentation broth of sp. ZS-1. Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) were used to detect and analyze components 1-5. The results showed that the molecular structures of components 1-5 corresponded one-to-one with streptomycin B, C, D, E and F, respectively.

[0102] The mass spectra of component 2 are as follows Figure 5 As shown in Figure 6, the H-NMR spectrum is as follows. 13 C-NMR such as Figure 7 As shown.

[0103] Mass spectra of component 4 are as follows Figure 8 As shown, H-NMR is as follows Figure 9 As shown, 13 C-NMR such as Figure 10 As shown.

[0104] The above results indicate that Streptomyces Streptomyces sp. ZS-1 can produce streptomycin, but the ratio of its components B, C, D, E, and F is different from that of *Streptomyces hainanense* var. *pulchella*. Streptomyces lavendulae There is a clear difference between var.hainanensis and UV-11.

[0105] Example 3 Streptomyces Streptomyces sp. ZS-1 fermentation level determination

[0106] Streptomyces Streptomyces sp. ZS-1, Streptomyces lilacinus Hainan variant ( Streptomyces lavendulae var. hainanensis) UV-11 and Streptomyces hainanensis ( Streptomyces lavendulae After being cultured in seed culture medium for 1 day, the 46# var.hainanensis were transferred to a 5L fermenter for 7 days of fermentation. Samples were then taken for testing of the content and bioavailability of the kasugamycin.

[0107] 1. Detection of the quality content of kasugamycin

[0108] Streptomyces were detected using high-performance liquid chromatography (HPLC). Streptomyces The fermentation broth of sp. ZS-1 was tested for streptomycin content, and the result was 16701 μg / mL.

[0109] The same method was used to treat *Streptomyces hainanensis* var. *lilacinus* (Hainan variety). Streptomyces lavendulaevar. hainanensis) UV-11, Streptomyces hainanensis ( Streptomyces lavendulae The fermentation broth of var.hainanensis new var.) 46# was tested for streptomycin content, and the results were 13819 μg / mL and 3668 μg / mL, respectively.

[0110] The results above show that Streptomyces Streptomyces The mass content of streptomycin in the fermentation broth of sp. ZS-1 was higher than that of Streptomyces hainanensis (a strain of Streptomyces lilacinus). Streptomyces lavendulae var. hainanensis) UV-11 was 20.9% higher than that of Streptomyces hainanensis (var. hainanensis). Streptomyces lavendulae var.hainanensis newvar.) 46# is 355.3% higher.

[0111] 2. Bioactivity testing

[0112] Streptomyces Streptomyces The biopotency of the fermentation broth of sp. ZS-1 was determined to be 23890 U / mL; the biopotency of *Streptomyces hainanense* var. *zinosa* was also measured. Streptomyces lavendulae var. hainanensis) UV-11, Streptomyces hainanensis ( Streptomyces lavendulae The biopotency of the fermentation broth of var. hainanensis (new var.) 46# was tested, and the results were 18402 U / mL and 4564 U / mL, respectively.

[0113] The results above show that Streptomyces Streptomyces The bioefficacy of ZS-1 fermentation broth compared to *Streptomyces hainanensis* (var. *zinosa*) Streptomyces lavendulae The UV-11 concentration of *Streptomyces hainanensis* var. *hainanensis* was 29.8% higher than that of *Streptomyces hainanensis* var. *hainanensis*. Streptomyces lavendulae var.hainanensis new var.) 46# higher than 423.4%.

[0114] The results above show that, regardless of whether the detection is based on the content of streptomycin or its biopotency, Streptomyces... Streptomyces The fermentation level of sp. ZS-1 was significantly higher than that of Streptomyces hainanensis (var. hainanensis). Streptomyces lavendulae var. hainanensis) UV-11 and Streptomyces hainanensis ( Streptomyces lavendulae var.hainanensis new var.) 46#.

[0115] Example 4 Streptomyces StreptomycesTest of antibacterial ability of fermentation product of Streptomyces sp. ZS-1

[0116] (1) Test of antibacterial ability of cucumber bacterial angular leaf spot

[0117] Take 1 mL of Streptomyces sp. ZS-1 fermentation broth prepared in Example 3, add 332 mL of water, and prepare a solution with a mass content of 50 μg / mL of mesamycin. Streptomyces sp. ZS-1 fermentation broth, add 332 mL of water, and prepare a solution with a mass content of 50 μg / mL of mesamycin;

[0118] Take 1 mL of Streptomyces sp. ZS-1 fermentation broth prepared in Example 3, add 332 mL of water, and prepare a solution with a mass content of 50 μg / mL of mesamycin. Streptomyces lavendulae var. hainanensis) UV-11 fermentation broth prepared in Example 3, add 275 mL of water, and prepare a solution with a mass content of 50 μg / mL of mesamycin.

[0119] Take 1 mL of Streptomyces sp. ZS-1 fermentation broth prepared in Example 3, add 332 mL of water, and prepare a solution with a mass content of 50 μg / mL of mesamycin. Streptomyces lavendulae var. hainanensis new var.) 46# fermentation broth, add 72 mL of water, and prepare a solution with a mass content of 50 μg / mL of mesamycin.

[0120] Take 200 mL of LB medium melted and cooled to 48-50°C, add 2 mL of 1x10 8 CFU / mL of cucumber bacterial angular leaf spot suspension, shake well, take an appropriate amount and pour into a culture dish (diameter 9 cm), stand and solidify to obtain a bacterial layer. Put 3 pieces of filter paper with a diameter of 6 mm, and drop 10 μL of the above solution on the filter paper, repeat 3 times. After 1 day of culture at 28°C, observe the size of the antibacterial ring produced by the fermentation broth, and the results are as follows:

[0121]

[0122] (2) Test of antibacterial ability of peanut Sclerotium blight

[0123] Take 0.60 mL of Streptomyces sp. ZS-1 fermentation broth prepared in Example 3, add 100 mL of melted PDA medium, and prepare a solution with a mass content of 100 μg / mL of mesamycin, pour into a culture dish to make a toxicity plate. Streptomyces Take 0.72 mL of Streptomyces sp. ZS-1 fermentation broth prepared in Example 3, add 100 mL of melted PDA medium, and prepare a solution with a mass content of 100 μg / mL of mesamycin, pour into a culture dish to make a toxicity plate.

[0124] Streptomyces lavendulae Take 0.72 mL of Streptomyces sp. ZS-1 fermentation broth prepared in Example 3, add 100 mL of melted PDA medium, and prepare a solution with a mass content of 100 μg / mL of mesamycin, pour into a culture dish to make a toxicity plate.

[0125] ​Take 2.73 mL of the *Streptomyces hainanense* strain prepared in Example 3 ( Streptomyces lavendulae Add 100 mL of melted PDA medium to the fermentation broth of var.hainanensis (new var.) 46# to prepare a solution with a kasugamycin content of 100 μg / mL, and pour it into a petri dish to make a toxicity plate.

[0126] Using a 6mm diameter punch, mycelial cakes were collected from the edge of activated peanut white mold fungus colonies (approximately 6 cm in diameter). These cakes were then inoculated onto the aforementioned virulence PDA plates, with a blank plate serving as a control. The inoculation was repeated three times, and the plates were incubated at 28℃ for 7 days. Colony growth in each group was investigated, and the inhibition rate was calculated. The formula was: Colony growth inhibition rate = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter) × 100%.

[0127] The growth status of each group of colonies is as follows: Figure 11 As shown, where A is Streptomyces. Streptomyces sp. ZS-1 fermentation broth, B is Streptomyces lilacinus Hainan variant ( Streptomyces lavendulae var. hainanensis) UV-11 fermentation broth, C is Streptomyces hainanensis (var. hainanensis) Streptomyces lavendulae (var. hainanensis new var.) 46# fermentation broth, D is the blank control. As can be seen from the figure, Streptomyces... Streptomyces sp. ZS-1 fermentation broth had the smallest colony diameter, and Streptomyces hainanensis (a variant of Streptomyces purpureus) was the largest. Streptomyces lavendulae var. hainanensis) UV-11 and Streptomyces hainanensis ( Streptomyces lavendulae The colony diameter of the fermentation broth (var.hainanensis new var.)46# was larger, while the diameter of the blank control was the largest.

[0128] The specific colony diameter and antibacterial effect statistics are shown in the table below:

[0129]

[0130] The above antibacterial test results show that Streptomyces Streptomyces The fermentation product of sp. ZS-1 has a strong inhibitory effect on bacterial angular leaf spot of cucumber, and its inhibitory effect is better than that of Streptomyces hainanensis var. hainanensis. Streptomyces lavendulae var. hainanensis) UV-11 and Streptomyces hainanensis ( Streptomyces lavendulae (var.hainanensis new var.) 46#. Additionally, the applicant unexpectedly discovered Streptomyces. Streptomycessp. ZS-1 also exhibits good inhibitory activity against *Streptomyces hainanense*, the causal agent of peanut white mold, while *Streptomyces lilacinus* Hainan varietal (… Streptomyces lavendulae var. hainanensis) UV-11 and Streptomyces hainanensis ( Streptomyces lavendulae The antibacterial ability of *Streptomyces hainanensis* var. (new var.) 46# is relatively weak, which is significantly different from the existing *Streptomyces* strains that can produce streptomycin. This indicates that *Streptomyces*... Streptomyces sp.ZS-1 has better antibacterial ability and a broader antibacterial spectrum.

[0131] Example 5 Streptomyces Streptomyces SCAR molecular marker analysis of sp. ZS-1

[0132] Streptomyces Streptomyces sp. ZS-1, Streptomyces lilacinus Hainan variant ( Streptomyces lavendulae var. hainanensis) UV-11, Streptomyces hainanensis ( Streptomyces lavendulae var.hainanensis new var.) 46#, Streptomyces Streptomyces sp. ZS-Ⅱ-D190515, Streptomyces MKL-2 ( Streptomyces microaureaus MKL-2), Streptomyces simonii ( Streptomyces caniferus NEAU6, Streptomyces Streptomyces Using genomic DNA from seven Streptomyces sp. 2356 strains as templates, random amplification was performed using 20 random primers (TGCCGAGCTG, AGTCAGCCAC, AATCGGGCTG, AGGGGTCTTG, GTTTCGCTCC, CATCCCCCCTG, CTGCTGGGAC, GTGAGGCGTC, CCGCATCTAC, GTGTGCCCCA, GGACCCAACC, TTGGCACGGG, AGCGCCATTG, GGTGCGGGAA, GTGACATGCC, TGCGCCCTTC, GGACTGCAGA, CTCACCGTCC, GGTGATCAGG, CTGAGACGGA). The amplified products were analyzed by agarose gel electrophoresis, revealing Streptomyces... Streptomyces sp. ZS-1 has unique bands that contain Streptomyces. Streptomyces The specific band of sp. ZS-1 was cut, recovered, and sequenced to obtain the sequence of the specific band.

[0133] According to the obtained specific band sequence, primers (forward primer as shown in SEQ ID NO: 2; reverse primer as shown in SEQ ID NO: 3) were designed, and the genomic DNA of the above 7 strains of Streptomyces was used as a template for specific amplification. The amplification products were analyzed by agarose gel electrophoresis, and the results are shown in Figure 1. Lanes 1-7 are Streptomyces sp. ZS-1, Streptomyces lavendulae var. hainanensis UV-11, Streptomyces lavendulae var. hainanensis new var. 46#, Streptomyces sp. ZS-II-D190515, Streptomyces parvus MKL-2, Streptomyces coelicolor NEAU6, and Streptomyces sp. 2356, respectively. As shown in Figure 1, only Streptomyces sp. ZS-1 can amplify a specific band, and the other 6 strains cannot amplify the specific band, verifying the specificity of the specific band sequence and the primers (SEQ ID NO: 2 and SEQ ID NO: 3) can specifically identify Streptomyces sp. ZS-1. Figure 12 Streptomyces Streptomyces lavendulae Streptomyces lavendulae Streptomyces Streptomyces microaureaus Streptomyces caniferus Streptomyces Streptomyces Streptomyces

[0134] The specific band obtained by specific amplification of Streptomyces sp. ZS-1 using SEQ ID NO: 2 and SEQ ID NO: 3 was recovered and sequenced, and the sequence of the specific band is shown in SEQ ID NO: 1. Streptomyces

[0135] The specific band sequence shown in SEQ ID NO: 1 was subjected to Blast comparison in GenBank, and no identical or similar sequences were found in all publicly available Streptomyces strains, which further indicates that the specific sequence shown in SEQ ID NO: 1 can be used to identify Streptomyces sp. ZS-1. Streptomyces

[0136] The above results show that the primers SEQ ID NO: 2 and SEQ ID NO: 3 are specific primers for identifying Streptomyces sp. ZS-1 strains, and the PCR amplification product, i.e., the sequence shown in SEQ ID NO: 1, is specific to Streptomyces sp. ZS-1. Streptomyces Streptomyces ​​​​​​​​​​​​​The SCAR molecular marker of Streptomyces sp. ZS-1. The primer of SEQ ID NO: 2 and SEQ ID NO: 3, and the specific band sequence (SEQ ID NO: 1) can quickly and effectively identify Streptomyces sp. ZS-1 Streptomyces Streptomyces sp. ZS-1.

[0137] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A mesomycin-producing strain, characterized by, The strain is Streptomyces sp. Streptomyces ZS-1, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 35482.

2. The mesimycin-producing strain according to claim 1, characterized by, The strain is Streptomyces sp. Streptomyces ZS-1, satisfying any one or a combination thereof: (1) the nucleotide sequence of the molecular marker is shown as SEQ ID NO: 1; (2) the specific band can be obtained by agarose gel electrophoresis analysis of the amplification product of the genomic DNA using the primers shown as SEQ ID NO: 2 and SEQ ID NO: 3; (3) the nucleotide sequence shown as SEQ ID NO: 1 is obtained by sequencing analysis of the amplification product of the genomic DNA using the primers shown as SEQ ID NO: 2 and SEQ ID NO:

3.

3. A molecular marker, characterized in that, The nucleotide sequence of the molecular marker is shown as SEQ ID NO:

1.

4. Use of the molecular marker of claim 3 in identifying the strain of any one of claims 1-2.

5. A pair of primers characterised in that, The nucleotide sequence of the forward primer is shown as SEQ ID NO: 2; The nucleotide sequence of the reverse primer is shown as SEQ ID NO:

3.

6. Use of the primer pair of claim 5 in identifying the strain of any one of claims 1-2.

7. A method for identifying the strain according to any one of claims 1 to 2, characterized in that, comprising the following steps: (1) sequencing the strain; (2) obtaining the sequence containing the specific molecular marker, which satisfies the following: 1) the nucleotide sequence is shown as SEQ ID NO:

1.

8. The method of claim 7, wherein, The sequencing is selected from: (1) genomic sequencing; and / or (2) sequencing of the PCR product.

9. The method of claim 8, wherein, The PCR product is obtained by amplification using the primers shown as SEQ ID NO: 2 and SEQ ID NO:

3.

10. Use of the strain of any one of claims 1-2 in preparing a pesticide product.

11. A pesticidal product, characterized in that It contains any one of the fermentation stock solution, fermentation diluent, fermentation concentrate of the strain of any one of claims 1-2.

12. The pesticidal product according to claim 11, characterized in that, The mycelismin content of the fermentation stock solution, fermentation diluent or fermentation concentrate is 10-300000 μg / mL.

13. The pesticidal product according to claim 11, characterized in that, The dosage form of the pesticide product is selected from any one of powder, granule, oil, seed treatment suspension.

14. The pesticidal product according to claim 11, characterized in that, The dosage form of the pesticide product is selected from slow release agent.

15. The pesticidal product of claim 11, wherein, The dosage form of the pesticide product is selected from suspension.

16. The pesticidal product of claim 11, wherein, The dosage form of the pesticide product is selected from suspension seed coating.

17. The pesticidal product of claim 11, wherein, The dosage form of the pesticide product is selected from any one of seed treatment dispersible powder, seed treatment soluble powder, seed treatment liquid, seed treatment emulsion, seed treatment microcapsule suspension.

18. The pesticidal product of claim 11, wherein, The dosage form of the pesticide product is selected from any one of large granule, fine granule, microgranule, microcapsule granule, wettable powder, oil dispersible powder, water dispersible granule, emulsion granule, effervescent granule, effervescent tablet, slow release block, slow release tube, slow release granule, soluble powder, soluble granule, soluble tablet, soluble liquid, water agent, soluble gel, film spreading oil, ultra-low volume liquid, ultra-low volume microcapsule suspension, emulsion oil, latex, dispersible liquid, paste, thick gum, water emulsion, oil emulsion, microemulsion, grease, microcapsule suspension, oil suspension, suspension emulsion.

19. Process for the preparation of a pesticide product according to any one of claims 11 to 18, characterized in that, The preparation method comprises the following steps: Fermentation using streptomyces Streptomyces sp. ZS-1 to obtain the pesticide product; The Streptomyces Streptomyces Streptomyces sp. ZS-1 is deposited at China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 35482.

20. The method of claim 19, wherein, The preparation method comprises the following steps: (1) Utilizing Streptomyces Streptomyces Sp. ZS-1 is fermented to obtain the fermentation stock solution, which is the pesticide product mentioned above; or The preparation method further comprises the following steps: (2) further processing the fermentation stock solution to obtain any one of fermentation dilute solution and fermentation concentrate solution, i.e. the pesticide product.

21. Use of the strain according to any one of claims 1-2, the pesticide product according to any one of claims 11-18 or obtained by the method according to any one of claims 19-20 in the prevention of plant diseases.

22. Use of the strain according to any one of claims 1-2, the pesticide product according to any one of claims 11-18 or obtained by the method according to any one of claims 19-20 in the preparation of a plant disease prevention agent.

Images